1. High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias
- Author
-
Vincent Van Deuren, Rob Lavigne, Johan Robben, and Sander Plessers
- Subjects
0301 basic medicine ,Phage display ,Chemistry, Multidisciplinary ,DIVERSITY ,RAPID DISCOVERY ,chemistry.chemical_compound ,SUBSTRATE ,INDEL Mutation ,INFECTION ,PEPTIDE ,Bacteriophages ,Biology (General) ,Oxford nanopore sequencing ,Spectroscopy ,M13 ,Illumina sequencing ,High-Throughput Nucleotide Sequencing ,General Medicine ,Computer Science Applications ,Chemistry ,Physical Sciences ,AlkB ,phage display ,FTO ,Life Sciences & Biomedicine ,Biochemistry & Molecular Biology ,PROTEINS ,QH301-705.5 ,Sequence analysis ,BACTERIOPHAGES ,Computational biology ,Biology ,Catalysis ,DNA sequencing ,Article ,Inorganic Chemistry ,03 medical and health sciences ,Peptide Library ,Physical and Theoretical Chemistry ,Indel ,QD1-999 ,Molecular Biology ,Illumina dye sequencing ,Science & Technology ,IDENTIFICATION ,030102 biochemistry & molecular biology ,Organic Chemistry ,RECOGNITION ,Genetic Variation ,Sequence Analysis, DNA ,amplification bias ,030104 developmental biology ,chemistry ,Nucleic acid ,Nanopore sequencing ,DNA ,parasitic enrichment - Abstract
The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins-AlkB and FTO-biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:11 ispartof: location:Switzerland status: published more...
- Published
- 2021
- Full Text
- View/download PDF